Rebecca Aft

Genome remodelling in a basal-like breast cancer metastasis and xenograft.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumour progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumour, a brain metastasis and a xenograft derived from the primary tumour. The metastasis contained two de novo mutations and a large deletion not present in the primary tumour, and was significantly enriched for 20 shared mutations. The xenograft retained all primary tumour mutations and displayed a mutation enrichment pattern that resembled the metastasis. Two overlapping large deletions, encompassing CTNNA1, were present in all three tumour samples. The differential mutation frequencies and structural variation patterns in metastasis and xenograft compared with the primary tumour indicate that secondary tumours may arise from a minority of cells within the primary tumour.

Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts

To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

Effect of zoledronic acid on disseminated tumour cells in women with locally advanced breast cancer: an open label, randomised, phase 2 trial

BACKGROUND Treatment with bisphosphonates decreases bone loss and can increase disease-free survival in patients with breast cancer. The aim of our study was to assess the effect of zoledronic acid on clearance of disseminated tumour cells (DTCs) from the bone marrow in women undergoing neoadjuvant chemotherapy for breast cancer. METHODS Patients were recruited for this open-label, phase 2 randomised trial between March 17, 2003, and May 19, 2006, at a single centre. Eligible patients had clinical stage II-III (> or = T2 and/or > or = N1) newly diagnosed breast cancer, Eastern Cooperative Oncology Group performance status of 0 or 1, and normal cardiac, renal, and liver function. 120 women were randomly assigned, using allocation concealment, to receive 4 mg zoledronic acid intravenously every 3 weeks (n=60), or no zoledronic acid (n=60), for 1 year concomitant with four cycles of neoadjuvant epirubicin (75 mg/m(2)) plus docetaxel (75 mg/m(2)) and two cycles of adjuvant epirubicin plus docetaxel. The primary endpoint was the number of patients with detectable DTCs at 3 months. Final analysis was done 1 year after the last patient was enrolled. Analyses were done for all patients with available data at 3 months. This study is registered with ClinicalTrials.gov, number NCT00242203. FINDINGS Of the 120 patients initially enrolled, one withdrew after signing consent and one patients baseline bone marrow was not available. Both of these patients were in the control group. At 3 months, 109 bone-marrow samples were available for analysis. In the zoledronic acid group, bone marrow was not collected from one patient because of disease progression, one patient was taken off study because of severe diarrhoea, and two patients had not consented at the time of surgery. In the control group, bone marrow was not collected from two patients because of disease progression, one patient withdrew consent, and three patients were not consented at the time of surgery. At baseline, DTCs were detected in 26 of 60 patients in the zoledronic acid group and 28 of 58 patients in the control group. At 3 months, 17 of 56 patients receiving zoledronic acid versus 25 of 53 patients who did not receive zoledronic acid had detectable DTCs (p=0.054). The most common grade 3-4 toxicities were infection (five of 60 patients in the zoledronic acid group and six of 59 in the control group) and thrombosis (five of 60 in the zoledronic acid and two of 59 in the control group). There was one documented case of osteonecrosis in the zoledronic acid group. INTERPRETATION Zoledronic acid administered with chemotherapy resulted in a decreased proportion of patients with DTCs detected in the bone marrow at the time of surgery. Our study supports the hypothesis that the antimetastatic effects of zoledronic acid may be through effects on DTCs. FUNDING Novartis Pharmaceuticals and Pfizer Inc.

Evaluation of 2-deoxy-D-glucose as a chemotherapeutic agent: mechanism of cell death

Nutrient deprivation has been shown to cause cancer cell death. To exploit nutrient deprivation as anti-cancer therapy, we investigated the effects of the anti-metabolite 2-deoxy-D-glucose on breast cancer cells in vitro. This compound has been shown to inhibit glucose metabolism. Treatment of human breast cancer cell lines with 2-deoxy-D-glucose results in cessation of cell growth in a dose dependent manner. Cell viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion assay and clonogenic survival are decreased with 2-deoxy-D-glucose treatment indicating that 2-deoxy-D-glucose causes breast cancer cell death. The cell death induced by 2-deoxy-D-glucose was found to be due to apoptosis as demonstrated by induction of caspase 3 activity and cleavage of poly (ADP-ribose) polymerase. Breast cancer cells treated with 2-deoxy-D-glucose express higher levels of Glut1 transporter protein as measured by Western blot analysis and have increased glucose uptake compared to non-treated breast cancer cells. From these results we conclude that 2-deoxy-D-glucose treatment causes death in human breast cancer cell lines by the activation of the apoptotic pathway. Our data suggest that breast cancer cells treated with 2-deoxy-D-glucose accelerate their own demise by initially expressing high levels of glucose transporter protein, which allows increased uptake of 2-deoxy-D-glucose, and subsequent induction of cell death. These data support the targeting of glucose metabolism as a site for chemotherapeutic intervention by agents such as 2-deoxy-D-glucose.

Targeting Chk1 in p53-deficient triple-negative breast cancer is therapeutically beneficial in human-in-mouse tumor models

Patients with triple-negative breast cancer (TNBC) - defined by lack of estrogen receptor and progesterone receptor expression as well as lack of human epidermal growth factor receptor 2 (HER2) amplification - have a poor prognosis. There is a need for targeted therapies to treat this condition. TNBCs frequently harbor mutations in TP53, resulting in loss of the G1 checkpoint and reliance on checkpoint kinase 1 (Chk1) to arrest cells in response to DNA damage. Previous studies have shown that inhibition of Chk1 in a p53-deficient background results in apoptosis [corrected] in response to DNA damage. We therefore tested whether inhibition of Chk1 could potentiate the cytotoxicity of the DNA damaging agent irinotecan in TNBC using xenotransplant tumor models. Tumor specimens from patients with TNBC were engrafted into humanized mammary fat pads of immunodeficient mice to create 3 independent human-in-mouse TNBC lines: 1 WT (WU-BC3) and 2 mutant for TP53 (WU-BC4 and WU-BC5). These lines were tested for their response to irinotecan and a Chk1 inhibitor (either UCN-01 or AZD7762), either as single agents or in combination. The combination therapy induced checkpoint bypass and apoptosis in WU-BC4 and WU-BC5, but not WU-BC3, tumors. Moreover, combination therapy inhibited tumor growth and prolonged survival of mice bearing the WU-BC4 line, but not the WU-BC3 line. In addition, knockdown of p53 sensitized WU-BC3 tumors to the combination therapy. These results demonstrate that p53 is a major determinant of how TNBCs respond to therapies that combine DNA damage with Chk1 inhibition.

Risk Factors for Surgical Site Infection after Major Breast Operation

BACKGROUND Understanding surgical site infection (SSI) risk factors after breast operation is essential to develop infection-prevention strategies and improve surgical outcomes. METHODS We performed a retrospective case-control study with subjects selected from a cohort of mastectomy, breast reconstruction, and reduction surgical patients between January 1998 and June 2002 at a university-affiliated hospital. SSI cases within 1 year after operation were identified using ICD-9-CM diagnosis codes for wound infection and complication or positive wound cultures, or both. Medical records of 57 patients with breast SSI and 268 randomly selected uninfected control patients were reviewed. Multivariate logistic regression was used to identify independent risk factors for SSI. RESULTS Significant independent risk factors for breast incisional SSI included insertion of a breast implant or tissue expander (odds ratio [OR] = 5.3; 95% CI, 2.5 to 11.1), suboptimal prophylactic antibiotic dosing (OR = 5.1; 95% CI, 2.5 to 10.2), transfusion (OR = 3.4; 95% CI, 1.3 to 9.0), mastectomy (OR = 3.3; 95% CI, 1.4 to 7.7), previous chest irradiation (OR = 2.8; 95% CI, 1.2 to 6.5), and current or recent smoking (OR = 2.1; 95% CI, 0.9 to 4.9). Local infiltration of an anesthetic agent was associated with substantially reduced odds of SSI (OR = 0.4; 95% CI, 0.1 to 0.9). CONCLUSIONS Suboptimal prophylactic antibiotic dosing is a potentially modifiable risk factor for SSI after breast operation. SSI risk was increased in patients undergoing mastectomy and in patients who had an implant or tissue expander placed during operation. This information can be used to develop a specific risk stratification index to predict SSI and infection-preventive strategies tailored for breast surgery patients.

Isolation and molecular profiling of bone marrow micrometastases identifies TWIST1 as a marker of early tumor relapse in breast cancer patients.

Purpose: Micrometastatic cells detected in the bone marrow have prognostic significance in breast cancer. These cells are heterogeneous and likely do not exhibit uniform biological behavior. To understand the molecular diversity of disseminated cancer cells that reside in bone marrow, we enriched this cell population and did global gene expression profiling in the context of a prospective clinical trial involving women with clinical stage II/III breast cancer undergoing neoadjuvant chemotherapy. Experimental Design: Enrichment of TACSTD1 (EpCAM)–expressing cells from bone marrow of breast cancer patients was achieved using immunomagnetic beads. Gene expression profiles were compared between enriched cell populations and whole bone marrow from 5 normal volunteers and 23 breast cancer patients after neoadjuvant chemotherapy treatment. Enriched cells from bone marrow samples of breast cancer patients before treatment or at 1 year follow-up were also analyzed (total of 87 data sets). The expression of transcripts specifically detected in enriched cell populations from breast cancer patients was correlated with 1-year clinical outcome using quantitative reverse transcription-PCR in an independent cohort of bone marrow samples. Results: Analysis of EpCAM-enriched bone marrow cells revealed specific expression of a subgroup of transcripts, including the metastasis regulator, TWIST1. Most transcripts identified, including TWIST1, were not expressed in enriched populations of bone marrow from normal volunteers, suggesting that this expression profile reflects a signature of breast cancer bone marrow micrometastases that persist after chemotherapy. In an independent set of bone marrow samples obtained before any treatment, TWIST1 expression correlated with early disease relapse. Conclusions: Disseminated breast cancer cells present in bone marrow after chemotherapy possess unique transcriptional signatures. Genes whose expression is overrepresented in these cell populations, such as TWIST1, may prove to be excellent markers of early distant relapse in breast cancer patients.

Temporal and spatial cooperation of Snail1 and Twist1 during epithelial-mesenchymal transition predicts for human breast cancer recurrence.

Epithelial–mesenchymal transition (EMT) is a normal developmental program that is considered to also play an important role in cancer metastasis. Ultimate inducers of EMT are transcriptional repressors that individually can induce experimental EMT, yet in many cells, particularly cancer cells, multiple inducers are expressed simultaneously. Why, and if, and how they interact to regulate EMT is unanswered. Using RNA interference technology to affect protein knockdown and avoid potential overexpression artifact coupled with transient TGFβ treatment to better mimic in vivo conditions we show, in both nontumorigenic and tumorigenic epithelial cancer cells, that Snail1 is uniquely required for EMT initiation, whereas Twist1 is required to maintain late EMT. Twist1, present in resting epithelial cells, is dispensable for EMT initiation. Mechanistically, in response to transient TGFβ treatment, transient Snail1 expression represses Twist1 transcription directly, which is subsequently upregulated, as Snail1 levels decrease, to sustain E-cadherin downregulation and growth arrest of EMT. Persistent Twist1 expression is associated with a p38 and extracellular signal–regulated kinase signal feedback loop that sustains growth-inhibitory signals characteristic of quiescent micrometastatic tumors. This Snail1–Twist1 temporal and spatial cooperation was also observed in vivo during human breast cancer progression to metastasis. Twist1 level, but not Snail1 level, and Twist1:Snail1 ratio in disseminated micrometastatic bone marrow tumor cells was found to correlate with survival and treatment resistance and is highly predictive of metastatic or recurrent disease. Mol Cancer Res; 9(12); 1644–57. ©2011 AACR.

IL-12p70–producing patient DC vaccine elicits Tc1-polarized immunity

BACKGROUND Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients, but dose-limiting toxicities have hindered its incorporation in vaccine formulations. Here, we report on the immunological and clinical outcomes upon vaccination with CD40L/IFN-γ-matured, IL-12p70-producing DCs. METHODS 7 HLA-A*0201+ newly diagnosed stage IV melanoma patients were immunized against the gp100 melanoma antigen using autologous peptide-pulsed, CD40L/IFN-γ-matured DCs. PBMCs were taken weekly for immune monitoring by tetramer analysis and functional assays. CT imaging was performed at baseline, week 9, and week 18 for clinical assessment using RECIST. RESULTS 6 of 7 treated patients developed sustained T cell immunity to all 3 melanoma gp100 antigen-derived peptides. 3 of the 6 immunological responders developed confirmed clinical responses (1 complete remission >4 years, 2 partial response). Importantly, DC vaccine-derived IL-12p70 levels positively correlated with time to progression (P = 0.019, log-rank), as did T-cytotoxic 1 (Tc1) immunity, as assessed by IFN-γ/IL-13 and IFN-γ/IL-5 ratios (P = 0.035 and P = 0.030, respectively, log-rank). In contrast, a pathway-specific defect in IL-12p35 transcription was identified upon CD40L/IFN-γ activation in clinical nonresponder patient DCs, and gp100-specific T cells from these patients displayed a Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ activation protocol corrected the IL-12p70 production defect in DCs derived from clinical nonresponder patients. CONCLUSION These findings underscore the essential role of IL-12p70 in the development of therapeutic type 1 antigen-specific CD8+ T cell immunity in humans with cancer.

Patient and tumor characteristics associated with increased mortality in young women (≤40 years) with breast cancer

The goal of the current study is to identify predictors responsible for mortality disparities between young (≤40 years) and older (>40 years) women with breast cancer.